Angiogenic factors have been defined as proteins which possess a variety of properties, namely the ability to (1) increase the rate of endothelial cell proliferation; (2) increase endothelial cell protease synthesis; (3) stimulate endothelial cell migration toward the protein location; and (4) cause in vivo capillary proliferation. In particular, it has been observed that substances classified as angiogenic factors can be mitogenic by affecting DNA synthesis in endothelial cells, thus increasing the rate of endothelial cell proliferation and the rate at which new blood vessels are formed.
Interrelated with this property is the ability of angiogenic factors to increase protease synthesis by endothelial cells. These proteases include plasminogen activator (PA) and collagenase. Specifically, the angiogenic factors are able to stimulate synthesis of PA and latent collagenase where the PA can convert zymogen plasmin into active plasmin, a protease of wide specificity, which in turn can convert latent collagenase into active collagenase. These two proteases, active plasmin and active collagenase, are capable of degrading most of the proteins in surrounding tissues, thus allowing increased invasion of various tissues, such as capillary endothelial cells. Moreover, angiogenic factors are chemotactic for certain cells, particularly capillary endothelial cells, i.e. they induce these cells to migrate toward the angiogenic factor.
With these properties in mind, it has been postulated that the isolation of an angiogenic factor would allow creation of a therapeutic substance capable of increasing the blood supply to an organ. For instance, subsequent to certain myocardial infarctions it would be desirable to stimulate regeneration of the blood supply to the heart interrupted as a result of the infarction or to stimulate re-growth of vessels in chronic obstructions. In addition, the use of an angiogenic factor may stimulate healing in decubitus ulcers, surgical incisions and slowly healing wounds, particularly in geriatric and diabetic patients. Moreover, the application of this material to burns may improve the rate and degree of healing. Therefore, a purified angiogenic factor suitable for therapeutic applications in humans has been sought. Additionally, some scientists believe that study of a substance capable of stimulating blood vessel growth may lead to processes for which the blood supply to a cancerous tumor might be inhibited, thus starving the cancer.
Previously, although a class of proteins had been identified which have been referred to as "angiogenic factors," these proteins were primarily isolated from non-human sources. It is believed that angiogenic factors isolated from non-human sources would not be suitable for use as therapeutic agents in humans due to the potential for adverse immunological reaction in response to a foreign protein. Moreover, it had not been demonstrated whether these non-human proteins individually possessed the four identified properties of an angiogenic factor identified above or whether the observed properties were attributable to the interactions between a combination of proteins.
Indeed, various proteins which have been found to have endothelial cell mitogenic properties have been divided into two classes: endothelial cell growth factor-like molecules which are eluted from heparin-Sepharose with 1 M NaCl and which have an acidic pI; and fibroblast growth factor-like molecules which bind more strongly to heparin-Sepharose and which have a basic pI. In addition, the present inventors believe that there is a third species of angiogenic factor, that described as "angiogenin" in papers recently published by Vallee et al. of Harvard Medical School, in Biochemistry, 1985, Vol. 24, pgs. 5480-5499. It is believed that angiogenin, while possessing some properties of a true angiogenic factor, is a distinct species in that it lacks mitogenic properties.
In the face of this patchwork of research, the present inventors sought and discovered a human angiogenic factor, classifiable as an FGF.sub.basic, which is substantially homologous to that isolatable from human placental tissue, which, in a single molecule, has the above-identified properties, i.e., is mitogenic, chemotactic, and capable of stimulating protease synthesis as well as capable of causing in vivo capillary proliferation. Furthermore, the present inventors sought to isolate this angiogenic factor in a substantially purified form from human placental tissues. The amino acid sequence of this isolated angiogenic factor has now been determined. It is believed that the determination of this amino acid sequence will allow identification of DNA probes for use in and obtaining genomic or cDNA sequences useful in recombinant-DNA methods for the synthesis of angiogenic factors.